Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Although adrenergic receptors (ARs) bind the same endogenous catecholamines (epinephrine and norepinephrine, NE) their physiological as well as pharmacological specificity is markedly diverse. This diversity is due primarily to the existence of at least nine different proteins encoding three distinct adrenergic receptors types (xcex11, xcex12, and xcex2). These proteins belong to the super-family of G-protein coupled receptors, and are characterized by a single polypeptide chain which span the plasma membrane seven times, with an extracellular amino terminus, and a cytoplasmic carboxyl terminus. The molecular cloning of three genes encoding xcex11-ARs supports the existence of pharmacologically and anatomically distinct xcex11-receptor subtypes. The xcex11b-receptor was originally cloned from a hamster smooth muscle cell line cDNA library, and encodes a 515 a.a. peptide that shows 42-47% homology with other ARs. The message for the xcex11b-receptor is abundant in rat liver, heart, cerebral cortex and kidney, and its gene was localized to human chromosome 5 (4). A second cDNA clone from a bovine brain library was found which encoded a 466-residue polypeptide with 72% homology to the xcex11b-AR gene. It was further distinguished from xcex11b by the finding that its expression was restricted to human hippocampus, and by its localization to human chromosome 8 and it has been designated as the xcex11c-AR (20). The cloning of an xcex11a-AR has been reported recently. This gene, isolated from a rat brain cDNA library, encodes a 560-residue polypeptide that shows 73% homology with the hamster xcex11b-receptor. The message for this subtype is abundant in rat vas deferens, aorta, cerebral cortex and hippocampus, and its gene has been localized to human chromosome 5 (12).
Pharmacological studies have demonstrated the existence of two xcex11-adrenergic receptor subtypes. The studies of xcex11-AR-mediated responses in vascular tissue suggested the possible existence of receptor subtypes, based on the potency and efficacy of adrenergic agonists, as well as differential sensitivity of xcex11 receptor-mediated responses to extracellular calcium and calcium channel blockers (6, 24). Although radioligand binding studies of brain xcex11-ARs with either [3H]WB4101 and [3H]prazosin showed good agreement with the potency of xcex1-adrenergic antagonists on vascular responses (23, 10), subsequent binding studies of rat brain xcex11-ARs provided strong evidence for the existence of receptor heterogeneity, based on the relative affinities for prazosin and WB4101 (15). These observations were supported by the finding that chloroethylclonidine (CEC) inactivated 50% of the xcex11 sites from rat cerebral cortex and 80% of the binding sites from liver or spleen (xcex11b), but did not inactivate xcex11-receptors from the hippocampus or vas deferens (xcex11a) (14). Taken together, these results suggested a classification of the xcex11a-subtype as high affinity for WB4101 and insensitive to alkylation by CEC, and xcex11b-subtype as 10 to 20 fold lower affinity for WB4101, but sensitive to inactivation by CEC. Consistent with this evidence the transfection of the hamster xcex11b gene into COS-7 cells induced the expression of an xcex11-receptor with high affinity for WB4101, 95% of which could be inactivated by CEC. Conversely, upon expression of the rat xcex11a receptor gene in COS-7 cells, it showed a 10-fold higher affinity for WB4101 than the xcex11b-receptor, and the binding site was resistant to inactivation by CEC. The existence of the xcex11c receptor was not predicted from pharmacological data and upon expression it showed 16 and 30 fold higher affinity for WB4101 and phentolamine respectively, than the xcex11b-receptor and was partially inactivated (65%) by CEC.
Molecular cloning and pharmacological studies have demonstrated the existence of at least three xcex11-adrenergic receptor subtypes. However, it is not clear whether the pharmacological properties of these three cognates might be due also to species differences. This caveat is particularly relevant in the case of the bovine xcex11c receptor, due to its restricted species and tissue expression. The cloning and expression of the human xcex11 adrenergic receptors will allow the further characterization of the pharmacology of the individual human xcex11 receptor subtypes.
This invention provides and isolated nucleic acid molecule encoding a human xcex11 adrenergic receptor. This invention further provides an isolated nucleic acid molecule encoding a human xcex11a receptor. In one embodiment of this invention, the nucleic acid molecule comprises a plasmid pcEXV-xcex11a. This invention also provides an isolated nucleic acid molecule encoding a human xcex11b receptor. In one embodiment of this invention, the nucleic acid molecule comprises a plasmid pcEXV-xcex11b. This invention further provides an isolated nucleic acid molecule encoding a human xcex11c receptor. In one embodiment of this invention, the nucleic acid molecule comprises a plasmid pcEXV-xcex11c.
This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human xcex11a receptor, adapted for expression in a bacterial, a yeast cell, or a mammalian cell which additionally comprise regulatory elements necessary for expression of the DNA in the bacteria, yeast or mammalian cells so located relative to the DNA encoding the human xcex11a receptor as to permit expression thereof. This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human xcex11b receptor, adapted for expression in a bacterial, a yeast cell, or a mammalian cell which additionally comprise regulatory elements necessary for expression of the DNA in the bacteria, yeast or mammalian cells so located relative to the DNA encoding the human xcex11b receptor as to permit expression thereof. This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human xcex11c receptor, adapted for expression in a bacterial, a yeast cell, or a mammalian cell which additionally comprise regulatory elements necessary for expression of the DNA in the bacteria, yeast or mammalian cells so located relative to the DNA encoding the human xcex11c receptor as to permit expression thereof.
This invention provides a mammalian cell comprising a DNA molecule encoding a human xcex11a receptor. This invention also provides a mammalian cell comprising a DNA molecule encoding a human xcex11b receptor. This invention also provides a mammalian cell comprising a DNA molecule encoding a human xcex11c receptor.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human xcex11a receptor. This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human xcex11b receptor. This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human xcex11c receptor.
This invention provides an antisense oligonucleotide having a sequence capable of specifically binding to any sequences of an mRNA molecule encoding a human xcex11a receptor so as to prevent translation of the mRNA molecule. This invention provides an antisense oligonucleotide having a sequence capable of specifically binding to any sequences of an mRNA molecule encoding a human xcex11b receptor so as to prevent translation of the mRNA molecule. This invention provides an antisense oligonucleotide having a sequence capable of specifically binding to any sequences of an mRNA molecule encoding a human xcex11c receptor so as to prevent translation of the mRNA molecule.
This invention provides method for detecting expression of a specific human xcex11 adrenergic receptor, which comprises obtaining RNA from cells or tissue, contacting the RNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human xcex11 receptor under hybridizing conditions, detecting the presence of any mRNA hybridized to the probe, the presence of mRNA hybridized to the probe indicating expression of the specific human xcex11 adrenergic receptor, and thereby detecting the expression of the specific human xcex11 adrenergic receptor.
This invention provides a method for detecting the expression of a specific human xcex11 adrenergic receptor in a cell or tissue by in situ hybridization which comprises, contacting the cell or tissue with a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human xcex11 receptor under hybridizing conditions, detecting the presence of any mRNA hybridized to the probe, the presence of mRNA hybridized to the probe indicating expression of the specific human xcex11 adrenergic receptor, and thereby detecting the expression of the specific human xcex11 adrenergic receptor.
This invention provides a method for isolating a nucleic acid molecule encoding a receptor by nucleic acid sequence homology using a nucleic acid probe, the sequence of which is derived from the nucleic acid sequence encoding a human al adrenergic receptor.
This invention provides a method for isolating a nucleic acid molecule encoding a human xcex11 adrenergic receptor which comprises the use of the polymerase chain reaction and oligonucleotide primers, the sequence of which are derived from the nucleic acid sequence encoding a human al adrenergic receptor.
This invention provides a method for isolating a human xcex11 adrenergic receptor protein which comprises inducing cells to express the human xcex11 adrenergic receptor protein, recovering the human xcex11 adrenergic receptor from the resulting cells, and purifying the human xcex11 adrenergic receptor so recovered.
This invention provides an antibody to the human xcex11a adrenergic receptor. This invention also provides an antibody to the human xcex11b adrenergic receptor. This invention also provides an antibody to the human xcex11c adrenergic receptor.
A pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a human xcex11a adrenergic receptor and a pharmaceutically acceptable carrier is provided by this invention. A pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a human xcex11b adrenergic receptor and a pharmaceutically acceptable carrier is provided by this invention. A pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a human xcex11c adrenergic receptor and a pharmaceutically acceptable carrier is provided by this invention.
A pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of a human xcex11a adrenergic receptor and a pharmaceutically acceptable carrier is provided by this invention. A pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of a human xcex11b adrenergic receptor and a pharmaceutically acceptable carrier is provided by this invention. A pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of a human xcex11c adrenergic receptor and a pharmaceutically acceptable carrier is provided by this invention.
This invention provides a transgenic non-human mammal whose genome comprises a nucleic acid molecule encoding a human xcex11 adrenergic receptor, the DNA molecule so placed as to be transcribed into antisense mRNA complementary to mRNA encoding a human a, adrenergic receptor and which hybridizes to mRNA encoding a human xcex11 adrenergic receptor thereby reducing its translation.
This invention provides a method for determining the physiological effects of varying the levels of expression of a specific human al adrenergic receptor which comprises producing a transgenic non-human mammal whose levels of expression of a human xcex11 adrenergic receptor can be varied by use of an inducible promoter.
This invention provides method for determining the physiological effects of expressing varying levels of a specific human xcex11 adrenergic receptor which comprises producing a panel of transgenic non-human mammals each expressing a different amount of the human xcex11 adrenergic receptor.
This invention provides a method for determining whether a ligand not known to be capable of specifically binding to a human xcex11 adrenergic receptor can bind to a human xcex11 adrenergic receptor, which comprises contacting a mammalian cell comprising a plasmid adapted for expression in a mammalian cell which further comprises a DNA molecule which expresses a human xcex11 adrenergic receptor on the cell surface with the ligand under conditions permitting binding of ligands known to bind to a human xcex11 adrenergic receptor, detecting the presence of any ligand bound to the human xcex11 adrenergic receptor, the presence of bound ligand thereby determining that the ligand binds to the human xcex11 adrenergic receptor.
This invention provides a method for screening drugs to identify drugs which interact with, and specifically bind to, a human xcex11 adrenergic receptor on the surface of a cell, which comprises contacting a mammalian cell which comprises a plasmid adapted for expression in a mammalian cell which further comprises a DNA molecule which expresses a human xcex11 adrenergic receptor on the cell surface with a plurality of drugs, determining those drugs which bind to the human xcex11 adrenergic receptor expressed on the cell surface of the mammalian cell, and thereby identifying drugs which interact with, and bind to, the human xcex11 adrenergic receptor.
This invention provides a method for identifying a ligand which binds to and activates or blocks the activation of, a human xcex11 adrenergic receptor expressed on the surface of a cell, which comprises contacting a mammalian cell which comprises a plasmid adapted for expression in a mammalian cell which further comprises a DNA molecule which expresses a human xcex11 adrenergic receptor on the cell surface with the ligand, determining whether the ligand binds to and activates or blocks the activation of the receptor using a bioassay such as a second messenger assays.
This invention also provides a method for identifying a ligand which is capable of binding to and activating or inhibiting a human xcex11 adrenergic receptor, which comprises contacting a mammalian cell, wherein the membrane lipids have been labelled by prior incubation with a labelled lipid precursor molecule, the mammalian cell comprising a plasmid adapted for expression in a mammalian cell which further comprises a DNA molecule which expresses a human xcex11 adrenergic receptor with the ligand and identifying an inositol phosphate metabolite released from the membrane lipid as a result of ligand binding to and activating an xcex11 adrenergic receptor.
This invention also provides a method for identifying a ligand that is capable of binding to and activating or inhibiting a human xcex11 adrenergic receptor, wherein the binding of ligand to the adrenergic receptor results in a physiological response, which comprises contacting a mammalian cell which comprises a plasmid adapted for expression in a mammalian cell which further comprises a DNA molecule which expresses a human xcex11 adrenergic receptor with a calcium sensitive fluorescent indicator, removing the indicator that has not been taken up by the cell, contacting the cells with the ligand and identifying an increase or decrease in intracellular Ca+2 as a result of ligand binding to and activating or inhibiting xcex11 adrenergic receptor activity.
This invention provides a method for detecting the presence of a human xcex11a adrenergic receptor on the surface of a cell, which comprises contacting the cell with an antibody to human xcex11a adrenergic receptor under conditions which permit binding of the antibody to the receptor, detecting the presence of any of the antibody bound to the human xcex11a adrenergic receptor and thereby the presence of a human xcex11a adrenergic receptor on the surface of the cell.
This invention provides a method for detecting the presence of a human xcex11b adrenergic receptor on the surface of a cell, which comprises contacting the cell with an antibody to human xcex11b adrenergic receptor under conditions which permit binding of the antibody to the receptor, detecting the presence of any of the antibody bound to the human xcex11b adrenergic receptor and thereby the presence of a human xcex11b adrenergic receptor on the surface of the cell.
This invention provides a method for detecting the presence of a human xcex11c adrenergic receptor on the surface of a cell, which comprises contacting the cell with an antibody to human xcex11c adrenergic receptor under conditions which permit binding of the antibody to the receptor, detecting the presence of any of the antibody bound to the human xcex11c adrenergic receptor and thereby the presence of a human xcex11c adrenergic receptor on the surface of the cell.
This invention provides a method of treating an abnormal condition related to an excess of activity of a human xcex11 adrenergic receptor subtype, which comprises administering an amount of a pharmaceutical composition effective to reduce xcex11 adrenergic activity as a result of naturally occurring substrate binding to and activating a specific xcex11 adrenergic receptor.
This invention provides a method for treating abnormalities which are alleviated by an increase in the activity of a specific human xcex11 adrenergic receptor, which comprises administering a patient an amount of a pharmaceutical composition effective to increase the activity of the specific human xcex11 adrenergic receptor thereby. alleviating abnormalities resulting from abnormally low receptor activity.
This invention provides a method for diagnosing a disorder or a predisposition to a disorder associated with the expression of a specific human xcex11 adrenergic receptor allele which comprises: a.) obtaining DNA from subjects suffering from a disorder; b.) performing a restriction digest of the DNA with a panel of restriction enzymes; c.) electrophoretically separating the resulting DNA fragments on a sizing gel; d.) contacting the gel with a nucleic acid probe labelled with a detectable marker and which hybridizes to the nucleic acid encoding a specific human xcex11 adrenergic receptor; e.) detecting the labelled bands which have hybridized to the DNA encoding the specific xcex11 adrenergic receptor labelled with the detectable marker to create a unique band pattern specific to the DNA of subjects suffering with the disorder; f.) preparing DNA for diagnosis by steps a-e; g.)comparing the unique band pattern specific to the DNA of patients suffering from the disorder from step e and DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.
This invention provides a method for identifying a substance capable of alleviating the abnormalities resulting from overexpression of a specific human xcex11 adrenergic receptor which comprises administering a substance to the transgenic non-human mammal comprising the DNA encoding a specific xcex11 adrenergic receptor and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of overexpression of the human xcex11 adrenergic receptor subtype.
This invention provides a method for identifying a substance capable of alleviating the abnormalities resulting from underexpression of a human xcex11 adrenergic receptor subtype, which comprises administering a substance to a non-human transgenic mammal which is expressing a human xcex11 adrenergic receptor incapable of receptor activity or is underexpressing the human xcex11 adrenergic receptor subtype, and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of underexpression of a human xcex11 adrenergic receptor subtype.
This invention provides a method of treating abnormalities in a subject, wherein the abnormality is alleviated by the reduced expression of a human xcex11 adrenergic receptor subtype which comprises administering to a subject an effective amount of the pharmaceutical composition effective to reduce expression of a specific xcex11 adrenergic receptor subtype.
This invention provides a method of treating abnormalities resulting from underexpression of a human xcex11 adrenergic receptor which comprises administering to a subject an amount of a pharmaceutical composition effective to alleviate abnormalities resulting from underexpression of the specific human xcex11 adrenergic receptor.